The major objectives of the proposed research are to reveal the mechanisms by which gene regulatory network (GRN) architecture ensures a constant output in response to genetic variation and to mechanistically dissect the processes that convert embryonic cells from multipotentiality to a committed state of differentiation. The well-described GRN that directs specification and differentiation of the endoderm during C. elegans embryogenesis will be used to investigate these problems. This GRN is initiated by the combined action of a maternal transcription factor, SKN-1, and a triply redundant Wnt, MAPK, and src signaling system. Analysis of 97 C. elegans wild isolates (isotypes), each with a unique haplotype, revealed dramatic variation in requirements for SKN-1 and MOM-2/Wnt in endoderm formation, allowing comprehensive dissection of genomic changes in GRN action. Further, we found that a novel Notch signaling system establishes a memory state in the early embryo that activates the embryonic multipotentiality commitment transition (MCT) and prevents cells from being reprogrammed by components of the endoderm GRN later in development. We will build on these preliminary findings to reveal mechanisms of genetic and developmental plasticity in the endoderm GRN through three specific aims. In Aim 1, we will characterize the molecular and genetic basis for broad variation seen among the C. elegans isotypes in the requirement for SKN-1 and Wnt signaling by identifying the relevant loci and their interactions via genome-wide association studies and QTL analysis. We will quantify expression differences in components of the endoderm GRN in selected strains with the goal of understanding how the genotypic changes alter flux, and allow for plasticity, in the network. In Aim 2, we will investigate the acton of the Notch signaling system and two novel secreted Notch ligands, DSL-1 and -3, in regulation of developmental plasticity and the timing of onset of the MCT. We will evaluate the hypotheses that this signaling system functions autonomously within the lineage of the major ectoblast, the AB cell, to regulate the MCT by the action of diffusible secreted molecules and that it acts on the MCT by regulating chromatin condensation. In Aim 3, we will perform RNAi-based screens to identify the comprehensive set of genes required for regulating developmental plasticity during embryogenesis. We will analyze the lineage, regional, and temporal specificity of genes required for timely execution of the MCT, assess the breadth of action of the genes in preventing alternative programs of cell type differentiation, and evaluate the molecular pathways through which the genes function. Findings from this research may lead to a better understanding of the processes required to generate new replacement tissues and organs in regenerative medicine. They will also serve as a paradigm for understanding the relationship between an individual's genotype and their responsiveness to pharmacological agents, thereby contributing to advances in personalized medicine.